The experiments proposed are aimed to characterize a new functional role for insulin and IGF receptors as transporters of their respective hormones across the endothelial cell barrier. To reach its target cells, circulating insulin has to traverse the vascular barrier which is lined by a continuous layer of endothelial cells. Endothelial cells were found to have receptors for insulin, IGF-I, and IGF-II with biochemical properties similar to those on other cells. These cells internalize and process insulin and IGFs differently than other cells since the majority of the internalized insulin and IGF-II are found to efflux from the endothelial cells without being degraded. Transport studies using specially designed cell culture chambers have demonstrated that insulin can be transported through the endothelial cells via an insulin-receptor related transcytosis pathway. In this proposal, we will characterize the intra-cellular sorting process and its regulation in endothelial cells. We will determine whether the structure of the hormone is important by comparing the cellular sorting of proinsulin, anti-insulin receptor antibodies, and IGF-I which bind to the insulin or IGF-I receptors. Insights into the role of the receptors will be provided by studying IGF-II which is structurally similar to insulin and IGF-I but binds to a different receptor. "Lysosomal" and cytoskeletal agents will be used to define the differences in the processing of various hormones. The effect of pH, calcium, and temperature will be studied due to their involvement in the dissociation of hormone-receptor complex which will be further explored to determine whether the receptors are transported with the hormone or are separately processed. The possibility that serine phosphorylation of hormone receptors might be a signal for the cell to internalize plasma membrane receptor will be studied by comparing the extent of serine phosphorylation of plasma membrane insulin and IGF receptors to their receptors from the intracellular pool. Since phorbol esters have been shown to alter serine phosphorylation on both insulin and IGF receptors, we will determine whether phorbol ester can affect the internalization and transport of these hormones in a similar manner. Possible interactions in the intracellular sorting and transport process will also be explored. Using the transport chamber, the dynamic interactions of IGF-I, IGF-II and their binding protein will be studied. Lastly, comparative experiments on the intracellular sorting and transcytosis process will be performed in endothelial cells derived from animals with and without diabetes of chronic duration.